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Effects of transition metal complexes on human lymphocyte blastogenesis in vitro
Pharmacological Research Communications, VoL 9, No. 2, 1977
EFFECTS OF TRANSITION METAL COMPLEXES ON HUIdAN LYMPHOCYTE BLASTOGENESIS IN VITRO
F. Bartoli Klugmann, B. Pani and G. Castellani Institutes of Pharmacology and Microbiology, University of Trieste and Fondazione C. & D. Callerio, Trieste, Italy. Received31 May 1976
SUMMARY The effect of two Platinum (II), two Rhodium (I), and two Ruthenium (II) complexes on the i_.nnvitro blastogenic response of human peripheral lymphocytes has been tested. All compounds except Ru(II)(DMSO) 2 ~-phen-Cl2 strongly inhibit the blastogenic response stimulated by mixed culture and by PHA. In the discussion of the results special emphasis is given to the pronounced difference between inhibition of PHA- and mixed culturestimulated lymphocytes shown by the two platinum compounds.
INTRODUCTION Recently, Gale made an important review (Gale, 1975) on cis-Pt(NH3) 2 C12 (II); an extensive report on the role of transition metal complexes th also appeared (SyzDosium 30 - Huston 1974). Among the interesting biological activities which Pt compounds have shown (Roberts et al., 1972; Shooter et al., 1972) is the inhibition of human lymphocyte blastogenic ~esponse stimulated by P~I (Phytohaemagglutinin) iqn vitr q (Gale, 1975; Howle et al., 1971). For this reason we thought first of confirming this activity of cis-PDD even on mixed culture. Likewise we tested the tran_~s isomer of PDD, two Rh compounds: Rh(I)COD 3,4,7,8 (CH3)4-phen + CI- and Rh(I)NBD
149
Pharmacological Research Communications, Voi. 9, No. 2, 1977
150
3,4,7,8 (CH3)4-phen ÷ CI-, and two Ru compounds: Ru(II)C12(DMSO) 4 and Ru(II)(DMSO) 2 R-phen-Cl 2 • We have chosen just these compounds due to the results obtained on their mutagenic activity on some bacterial strains (Monti-Bragadin et al., 1974; Monti-Bragadin induce prophage,
et al., 1975), through their ability to
to determine filamentous growth of Escherichia coli
(Monti-Bragadin et al., 1975) and due to the antitumour activity of Platinum and Rhodium compounds
(Giraldi et al., 1975; Rosenberg,
1973).
MATERIALAND METHODS The cis PDD isomer, the trans PDD isomer, the Rh compounds and the Ru compounds were prepared by Dr. Mestroni of the Chemical Department of our University. The antimitogenic activity of these compounds has been tested usins venous blood from presumably normal donors. The culture method used is that described by Pauly et al. (1973), which is the whole blood tecnique, with some modifications,
with the
final concentration of the blood being l:iO in MEM of Eagle. Cultures were incubated for five days. The blastogenic response was elicited A) by the mitogen PHA-M Difco (0,2 ml/lO ml blood suspension)
and B) by homologous blood.
The latest was a two-way culture obtained by adding diluted blood from each of two randomly selected unrelated donors with a ratio beinK i:I. The six compounds were dissolved in Dimethylsulfoxide
(DMSO), added
to the cultures on the fourth day= and were incubated for twenty four hours before testing. The controls contained only DMSO. The concentrations of Pt compounds were the same used in other experiments (Gale et al., 1971; Howle et al., 1971), and ranged from -6 -3 i0 to i0 M. The values of concentrations
of the Rh and Ru compounds were
Pharmacological Research CommunicaNons, Vol. 9, No. 2, 1977 deduced from
experiments
on the muta~enic and antibacterial activity;
for Rh compounds they were 10 -7,3 , 10 -7, 10 compounds,
151
~6,7
~6,4 , i0
M; for Ru
10 -4'6, 10 -4'3, 10 -4 , 10 -3'7 M.
The blastogenic response was determined by the extent of (6-3H) thymidine (Radiochemical Centre, Amersham - Buckinghamshire,
0,4/~Ci/ml
culture) uptake in the acid insoluble material as assessed by liquid scintillation countinE (Packard Model 2002) and the values were indicated as percent of cpm of the controls. Three separate experiments were done for each
compound
and every-
time all the samples were in duplicate. The differences between the highest and the lowest of the six values were ~ i0%. Therefore the points of the Eraphs represent the mean points of these intervals.
RESULTS AND DISCUSSION The two Pt compounds,
the two Rh compounds and one of the Ru
compounds (Ru(II)CI2(DMSO) 4 showed an inhibitory effect on the blastogenic response by PHA as well as by mixed culture as shown in fig. i. It is noteworthy that the tran__,ss-isomer of PDD, non-effective on any other biological system, ~ave, on the contrary,
the same result as
cis-PDD on the blastogenic response inhibition test. ~4oreover, the onhibitory effect of the Pt and Rh complexes was less evident on mixed cultures than on the PHA stimulated cultures. In contrast, the Ru complexes did not show appreciable difference in the inhibition activity on the two culture systems. Therefore we can confirm the results of literature for cis-PDD (Howle et al., 1971) but we would like to emphasize that even the trans-PDD is effective in this test. From the results obtained with the two Rh complexes and with one of Ruthenium complexes we can conclude that other transition metal complexes possess biological properties similar to those of Pt (Giraldiet
al., 1974). There are some important papers already published
Pharmacological Research Communications, Vol. 9, No. 2, 1977
152 A
B
60
60
4Q
40
20
20
0 0 D
(I) .J
"6
O
_
I0(
100
-S
-4
"- -b ~
m --
I
-3
•
C
lZ 0
I00
U
80
I00 t 80
Lu -r
IU.
0
40 20
~,,b
0
:E
40-
~%
i
7.3,
-7
20
• =, ~. -e.7
-
-6.4
. . . . . . . 7.3 "7
0
-6.7
-6.4
G.
U 14.
0
o~
E
F
I00
IOO
8Q
80
bO
60
40
40
20
20 ,,1,
0
.4.b
,
l
-4.3
,
I
,
-4
i
,i ,, -4.6
,
-3.7
i,
•
-4.3
-4
'
..3.7
LOG ,
-
CULTURE
IITIMULATEO
BY
liMA
~..-...~MIXEO
CULTURE
Figure 1 - Effects of six transition metal complexes on the "in vitro" blastogenic response of lymphocytes: A: Effect of cis-PDD B: Effect of tran,.s-PDD
C: E f f e c t o f Rh(I)COD-3,a,7,8(CH3)4Phen+Cl ÷
--
D: E f f e c t o f Rh(I)NBD-3,a,7,8(CH3)4Phen C1 E: E f f e c t o f Ru(IZ)CI2(DMSO) 4 F:
Effect
of
Ru(IZ)(DMSO)2-o'-phenCl 2
Pharmacological Research Communications, Vol. 9, No. 2, 1977
153
about the mechanism of action of cis-PDD describing its effect on bacterial as well as on eucariotic cells (Gale, 1975; Gale et al., 1971; Monti-Bragadin et al., 1974; Nonti-Bragadin et al., 1975). However, the mechanism of action of the other transition metal complexes iz not understood in detail, because of the lack of systematic investigations. Another noteworthy aspect is that the blastogenic response is inhibited quite differently by the two stimulating agents; a possible explanation of this fact is that PHA is a selective mytogen for T lymphocytes whilst in the mixed culture two different populationsl T and B lymphocytes are stimulated (Greaves et al., 1972; Mayry et al., 1972; Howle et al., 1973). Therefore we suppose that the PHA-stimulated culture being homogeneous is completely inhibited by Pt compounds. On the contrary the mixed culture seems to be less sensitive to the inhibitory effect, probably because the Pt compounds interact in a different way with the two populations. This hypothesis would be in agreement with the electrophoretic mobility data taken from the two populations which suggest that T cells have more negative charges on their surface than B cells (Greaves et al., 1972). These conclusions might be supported by Howle and Gale's papers (Gale, 1975; Howle et al., 1971) on the chemical transformation which these Pt compotu,ds must undergo to become biologically active. These authors propose a two-stage transformation in which the Pt compounds give rise to positively charged intermediates:
I
Pt(NH3) 2 C12
II
P t ( ~ 3 ) 2 Cl +
]k
Pt(NH3) 2 Cl + + CIg
Pt(NH3) ++ + Cl
Since T ceils have more negative charges on their surface they may interact better with the PDD intermediates. Such e f f e c t
may e x p l a i n
the lower inhibition in ~le mixed culture.
ACKNOWLEDGEMENTS
The a u t h o r s t h a n k Mr. C. Oamboz f o r h i s s k i l f u l
technical
assistance.
154
Pharmacological Research Communications, VoL 9, No 2, 1977
REFERENCES i
GALE G.R.
(1975) - Platinum compounds ~n. Antineoplastic and immunosuppressive agents I I E d . A.C. Sartorelli and D.G. Johns, Springer Verlag, p a g . 829.
GALE G.R., HOWLE J.A., WALKER E.M. jr. (1971) - Cancer Res. 31, 950. GIRALDI T., ZASSINOVICH G., MESTRONI G. (1974) - Chem. Biol. Interact. ~, 389. GREAVES M., JANOSSY G. (1972) - Transplant.
Rev. ii, 87.
HOWLE J.A., THOMSON M.S., STONE A.E., GALE G.R. Biol. Med. 137, 820.
(1971) - Proc. Soc. Exp.
MAYRY P., ANDERSON L.C., NORDLING S., VIROLAIMEN M. (1972) - Transplant. Rev. 12, 91. MONTI-BRAGADIN C., GIRALDI T., CANTINI M., ZASSINOVICH G., MESTRONI G. (1974) - FEBS Letters 43, 13. MONTI-BRAGADIN C., T~4ARO M., BANFI E. (1975) - Chem. Biol. Interact. l_!l, 4 6 9 . MONTI-BRAGADIN C., RAMANI L., SAMER L., MESTRONI G., ZASSINOVICH G. (1975) - Antimicrob. Ag. Chemother. Z, 825. P A U L Y J.L., SOKAL J.E., HAU T. (1973) - J. Lab. Clin. Med. 82, 500. ROBERTS J.J., PASCOE J.M.
(1972) - Nature 235, 282.
ROSENBERG B. (1973) - Naturwissenshafter 6~0, 99. SHOOTER K.V., HOWSE R., MENIFIELD R.K., ROBINS A.B. (1972) - Chim. Biol. Interactions ~, 289. SYMPOSIUM presented at the 30 th Southwest Regional Meeting of the American Chemical Society - Huston - Texas, December I0-II, 1974, Cancer Chem. Rep. Part I, Vol. 59, N. 3, May/June 1975.
EFFECTS OF TRANSITION METAL COMPLEXES ON HUIdAN LYMPHOCYTE BLASTOGENESIS IN VITRO
F. Bartoli Klugmann, B. Pani and G. Castellani Institutes of Pharmacology and Microbiology, University of Trieste and Fondazione C. & D. Callerio, Trieste, Italy. Received31 May 1976
SUMMARY The effect of two Platinum (II), two Rhodium (I), and two Ruthenium (II) complexes on the i_.nnvitro blastogenic response of human peripheral lymphocytes has been tested. All compounds except Ru(II)(DMSO) 2 ~-phen-Cl2 strongly inhibit the blastogenic response stimulated by mixed culture and by PHA. In the discussion of the results special emphasis is given to the pronounced difference between inhibition of PHA- and mixed culturestimulated lymphocytes shown by the two platinum compounds.
INTRODUCTION Recently, Gale made an important review (Gale, 1975) on cis-Pt(NH3) 2 C12 (II); an extensive report on the role of transition metal complexes th also appeared (SyzDosium 30 - Huston 1974). Among the interesting biological activities which Pt compounds have shown (Roberts et al., 1972; Shooter et al., 1972) is the inhibition of human lymphocyte blastogenic ~esponse stimulated by P~I (Phytohaemagglutinin) iqn vitr q (Gale, 1975; Howle et al., 1971). For this reason we thought first of confirming this activity of cis-PDD even on mixed culture. Likewise we tested the tran_~s isomer of PDD, two Rh compounds: Rh(I)COD 3,4,7,8 (CH3)4-phen + CI- and Rh(I)NBD
149
Pharmacological Research Communications, Voi. 9, No. 2, 1977
150
3,4,7,8 (CH3)4-phen ÷ CI-, and two Ru compounds: Ru(II)C12(DMSO) 4 and Ru(II)(DMSO) 2 R-phen-Cl 2 • We have chosen just these compounds due to the results obtained on their mutagenic activity on some bacterial strains (Monti-Bragadin et al., 1974; Monti-Bragadin induce prophage,
et al., 1975), through their ability to
to determine filamentous growth of Escherichia coli
(Monti-Bragadin et al., 1975) and due to the antitumour activity of Platinum and Rhodium compounds
(Giraldi et al., 1975; Rosenberg,
1973).
MATERIALAND METHODS The cis PDD isomer, the trans PDD isomer, the Rh compounds and the Ru compounds were prepared by Dr. Mestroni of the Chemical Department of our University. The antimitogenic activity of these compounds has been tested usins venous blood from presumably normal donors. The culture method used is that described by Pauly et al. (1973), which is the whole blood tecnique, with some modifications,
with the
final concentration of the blood being l:iO in MEM of Eagle. Cultures were incubated for five days. The blastogenic response was elicited A) by the mitogen PHA-M Difco (0,2 ml/lO ml blood suspension)
and B) by homologous blood.
The latest was a two-way culture obtained by adding diluted blood from each of two randomly selected unrelated donors with a ratio beinK i:I. The six compounds were dissolved in Dimethylsulfoxide
(DMSO), added
to the cultures on the fourth day= and were incubated for twenty four hours before testing. The controls contained only DMSO. The concentrations of Pt compounds were the same used in other experiments (Gale et al., 1971; Howle et al., 1971), and ranged from -6 -3 i0 to i0 M. The values of concentrations
of the Rh and Ru compounds were
Pharmacological Research CommunicaNons, Vol. 9, No. 2, 1977 deduced from
experiments
on the muta~enic and antibacterial activity;
for Rh compounds they were 10 -7,3 , 10 -7, 10 compounds,
151
~6,7
~6,4 , i0
M; for Ru
10 -4'6, 10 -4'3, 10 -4 , 10 -3'7 M.
The blastogenic response was determined by the extent of (6-3H) thymidine (Radiochemical Centre, Amersham - Buckinghamshire,
0,4/~Ci/ml
culture) uptake in the acid insoluble material as assessed by liquid scintillation countinE (Packard Model 2002) and the values were indicated as percent of cpm of the controls. Three separate experiments were done for each
compound
and every-
time all the samples were in duplicate. The differences between the highest and the lowest of the six values were ~ i0%. Therefore the points of the Eraphs represent the mean points of these intervals.
RESULTS AND DISCUSSION The two Pt compounds,
the two Rh compounds and one of the Ru
compounds (Ru(II)CI2(DMSO) 4 showed an inhibitory effect on the blastogenic response by PHA as well as by mixed culture as shown in fig. i. It is noteworthy that the tran__,ss-isomer of PDD, non-effective on any other biological system, ~ave, on the contrary,
the same result as
cis-PDD on the blastogenic response inhibition test. ~4oreover, the onhibitory effect of the Pt and Rh complexes was less evident on mixed cultures than on the PHA stimulated cultures. In contrast, the Ru complexes did not show appreciable difference in the inhibition activity on the two culture systems. Therefore we can confirm the results of literature for cis-PDD (Howle et al., 1971) but we would like to emphasize that even the trans-PDD is effective in this test. From the results obtained with the two Rh complexes and with one of Ruthenium complexes we can conclude that other transition metal complexes possess biological properties similar to those of Pt (Giraldiet
al., 1974). There are some important papers already published
Pharmacological Research Communications, Vol. 9, No. 2, 1977
152 A
B
60
60
4Q
40
20
20
0 0 D
(I) .J
"6
O
_
I0(
100
-S
-4
"- -b ~
m --
I
-3
•
C
lZ 0
I00
U
80
I00 t 80
Lu -r
IU.
0
40 20
~,,b
0
:E
40-
~%
i
7.3,
-7
20
• =, ~. -e.7
-
-6.4
. . . . . . . 7.3 "7
0
-6.7
-6.4
G.
U 14.
0
o~
E
F
I00
IOO
8Q
80
bO
60
40
40
20
20 ,,1,
0
.4.b
,
l
-4.3
,
I
,
-4
i
,i ,, -4.6
,
-3.7
i,
•
-4.3
-4
'
..3.7
LOG ,
-
CULTURE
IITIMULATEO
BY
liMA
~..-...~MIXEO
CULTURE
Figure 1 - Effects of six transition metal complexes on the "in vitro" blastogenic response of lymphocytes: A: Effect of cis-PDD B: Effect of tran,.s-PDD
C: E f f e c t o f Rh(I)COD-3,a,7,8(CH3)4Phen+Cl ÷
--
D: E f f e c t o f Rh(I)NBD-3,a,7,8(CH3)4Phen C1 E: E f f e c t o f Ru(IZ)CI2(DMSO) 4 F:
Effect
of
Ru(IZ)(DMSO)2-o'-phenCl 2
Pharmacological Research Communications, Vol. 9, No. 2, 1977
153
about the mechanism of action of cis-PDD describing its effect on bacterial as well as on eucariotic cells (Gale, 1975; Gale et al., 1971; Monti-Bragadin et al., 1974; Nonti-Bragadin et al., 1975). However, the mechanism of action of the other transition metal complexes iz not understood in detail, because of the lack of systematic investigations. Another noteworthy aspect is that the blastogenic response is inhibited quite differently by the two stimulating agents; a possible explanation of this fact is that PHA is a selective mytogen for T lymphocytes whilst in the mixed culture two different populationsl T and B lymphocytes are stimulated (Greaves et al., 1972; Mayry et al., 1972; Howle et al., 1973). Therefore we suppose that the PHA-stimulated culture being homogeneous is completely inhibited by Pt compounds. On the contrary the mixed culture seems to be less sensitive to the inhibitory effect, probably because the Pt compounds interact in a different way with the two populations. This hypothesis would be in agreement with the electrophoretic mobility data taken from the two populations which suggest that T cells have more negative charges on their surface than B cells (Greaves et al., 1972). These conclusions might be supported by Howle and Gale's papers (Gale, 1975; Howle et al., 1971) on the chemical transformation which these Pt compotu,ds must undergo to become biologically active. These authors propose a two-stage transformation in which the Pt compounds give rise to positively charged intermediates:
I
Pt(NH3) 2 C12
II
P t ( ~ 3 ) 2 Cl +
]k
Pt(NH3) 2 Cl + + CIg
Pt(NH3) ++ + Cl
Since T ceils have more negative charges on their surface they may interact better with the PDD intermediates. Such e f f e c t
may e x p l a i n
the lower inhibition in ~le mixed culture.
ACKNOWLEDGEMENTS
The a u t h o r s t h a n k Mr. C. Oamboz f o r h i s s k i l f u l
technical
assistance.
154
Pharmacological Research Communications, VoL 9, No 2, 1977
REFERENCES i
GALE G.R.
(1975) - Platinum compounds ~n. Antineoplastic and immunosuppressive agents I I E d . A.C. Sartorelli and D.G. Johns, Springer Verlag, p a g . 829.
GALE G.R., HOWLE J.A., WALKER E.M. jr. (1971) - Cancer Res. 31, 950. GIRALDI T., ZASSINOVICH G., MESTRONI G. (1974) - Chem. Biol. Interact. ~, 389. GREAVES M., JANOSSY G. (1972) - Transplant.
Rev. ii, 87.
HOWLE J.A., THOMSON M.S., STONE A.E., GALE G.R. Biol. Med. 137, 820.
(1971) - Proc. Soc. Exp.
MAYRY P., ANDERSON L.C., NORDLING S., VIROLAIMEN M. (1972) - Transplant. Rev. 12, 91. MONTI-BRAGADIN C., GIRALDI T., CANTINI M., ZASSINOVICH G., MESTRONI G. (1974) - FEBS Letters 43, 13. MONTI-BRAGADIN C., T~4ARO M., BANFI E. (1975) - Chem. Biol. Interact. l_!l, 4 6 9 . MONTI-BRAGADIN C., RAMANI L., SAMER L., MESTRONI G., ZASSINOVICH G. (1975) - Antimicrob. Ag. Chemother. Z, 825. P A U L Y J.L., SOKAL J.E., HAU T. (1973) - J. Lab. Clin. Med. 82, 500. ROBERTS J.J., PASCOE J.M.
(1972) - Nature 235, 282.
ROSENBERG B. (1973) - Naturwissenshafter 6~0, 99. SHOOTER K.V., HOWSE R., MENIFIELD R.K., ROBINS A.B. (1972) - Chim. Biol. Interactions ~, 289. SYMPOSIUM presented at the 30 th Southwest Regional Meeting of the American Chemical Society - Huston - Texas, December I0-II, 1974, Cancer Chem. Rep. Part I, Vol. 59, N. 3, May/June 1975.